Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency

Abstract

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

Figure 1. Mean (±SEM) mtDNA copy number in maturing and mature oocytes determined by real time PCR.

(a) BCB⁺ and (b) BCB⁻ maturing (0 hr and 22 hr) and immature (44 hr) and mature (44 hr) oocytes (n = 10 for each). Statistical analysis was performed using ANOVA. (c) Comparison between BCB⁺ and BCB⁻ MII oocytes performed by t-test.

Figure 2. Generation of mICSI-derived blastocysts.

(a) Localization of mitochondria following injection into BCB⁻ MII oocytes at 1 hr and 24 hr post insemination. Mitochondria endogenous to the oocyte are labeled with MitoTracker Deep Red (grey). The injected mitochondria are labeled with TMRM (red) and MitoTracker Green (green). The confocal z-stacks are displayed as maximum intensity projections (X40 magnification). (b) Transmission Electron Microscopy of MII oocytes, ICSI-1 hr, ICSI-24 hr, mICSI-1 hr, and mICSI-24 hr at 2500X, 5000X and 25,000X magnification. Abreviations include; LD = lipid droplet, V = vacuole, M = mitochondria. (c) DAPI staining of ICSI BCB⁺ and mICSI BCB⁻ blastocysts highlighting the presence of an inner cell mass. (d) Enlargement of a mICSI BCB⁻ blastocyst.

Figure 3. MtDNA copy number for preimplantation embryos.

(a) Mean (±SEM) mtDNA copy number for BCB⁺ IVF; (b) BCB⁺ ICSI; (c) BCB⁻ ICSI and (d)BCB⁻ mICSI-derived embryos determined by real time PCR (n = 5–10). (e) Mean (±SEM) mtDNA copy for each stage of development for embryos generated by IVF, ICSI and mICSI from BCB⁺ and BCB⁻ oocytes.

Figure 4. Global gene expression analysis of single blastocysts following microarray.

(a) PCA of microarray data from single blastocysts. Red, blue and brown points represent individual transcriptomes from ICSI BCB⁺ , ICSI BCB⁻ and mICSI BCB⁻ blastocysts, respectively. (b) Heat map of global gene expression following Pearson’s correlation to determine hierarchical clustering between blastocysts of each group. (c) Venn diagram representing differentially expressed genes between ICSI BCB⁺ and ICSI BCB⁻; mICSI BCB⁻ and ICSI BCB⁻; and mICSI BCB⁻ and ICSI BCB⁺ blastocysts following unpaired t-tests, with FC > 2 (abs) and significance of p < 0.01.